This proposal focuses on the CHC22 isoform of clathrin, which is preferentially expressed in human skeletal muscle. Recent work from the laboratory indicates that CHC22 is involved in intracellular sequestration of the GLUT4 glucose transporter. Upon insulin stimulation of skeletal muscle and adipocytes, GLUT4 is released from the GLUT4 storage compartment (GSC) in vesicles that fuse with the plasma membrane, where it can import glucose in response to acute metabolic needs. Thus GLUT4 membrane traffic is critical for glucose metabolism and its disruption is associated with type 2 diabetes. The implication of CHC22 clathrin in GLUT4 membrane traffic in humans is interesting because CHC22 is missing from mice and CHC22 expression may therefore account for some of the known differences between glucose metabolism by humans and mice. This proposal aims to characterize the role that CHC22 plays in GLUT4 transport, to determine the molecular mechanisms that control CHC22 function and to explore polymorphisms in CHC22 that would affect its function and thereby contribute to incidence of type 2 diabetes. For Aim 1, basic questions about CHC22-mediated membrane traffic of GLUT4 will be addressed, including molecular details of CHC22 function following insulin stimulation and its distribution in the muscle of diabetic patients. Experiments will compare GLUT4 membrane traffic in humans with its traffic in CHC22-transgenic mice. The presence and role of CHC22 in human adipocytes will also be investigated. For Aim 2, the factors needed for CHC22 to function properly will be established using two assays for its function. Genetic screens will be designed to re-create a functional pathway for CHC22 when it is introduced in mouse cells and to dissect CHC22 function in GSC formation. Aim 3 of this project is to characterize the biochemical properties of CHC22 that mediate its function. CHC22 protein will be purified from both tissue and recombinant sources to determine its morphology and whether it can assemble into macromolecular complexes. CHC22-associated proteins will be identified. Recombinant fragments of CHC22 protein will be expressed and their structure determined at crystallographic resolution. Aim 4 is to determine the distribution of CHC22 polymorphisms within human populations for correlation with incidence of type 2 diabetes. The functional features of common CHC22 variants will be characterized. In addition, phylogenetic analysis of CHC22 will assess whether its function in different species is related to nutrition habits. With these aims the overall goal of this proposal is to further characterize the role that CHC22 plays in GLUT4 transport in order to understand how human glucose metabolism is regulated. While studies of mice have provided major insights into pathways relevant to type 2 diabetes, CHC22 represents a missing species-specific element. The research proposed here will establish novel characteristics of human glucose homeostasis and will enable development of better animal models for studying its dysfunction in diabetes.